Positive pathogens in stool could predict the clinical outcomes of sepsis-associated acute kidney injury in critical ill patient

In this study, we sought to evaluate the influence of positive pathogens in stool (PPS) on clinical outcomes in critical ill patients with Sepsis-associated acute kidney injury (S-AKI) from intensive care unit. Our sample consisted of 7338 patients, of whom 752 (10.25%) had PPS. We found that the presence of Clostridium difficile (C. difficile) and protists in stool samples was correlated with survival during hospitalization, as well as 30-day and 90-day survival. Interestingly, there was no significant difference in overall survival and 30-day in-hospital survival between the PPS group and the negative pathogens in stool (NPS) control group. However, the cumulative incidence of 90-day infection-related mortality was significantly higher in the PPS group (53 vs. 48%, P = 0.022), particularly in patients with C. difficile in their stool specimens. After adjusting for propensity scores, the results also have statistical significance. These findings suggest that PPS may affect the 90-days survival outcomes of S-AKI, particularly in patients with C. difficile and protists in their stool samples. Further research is warranted to further explore these associations.


The impact of PPS on sepsis AKI
There was no statistical difference between the SOFA and GCS scores, indicating a similar level of critical illness in both groups of patients (Table 1, P > 0.05).The median length of hospital stays for the NPS and PPS groups was 17 days (range: 10-29) and 22 days (range: 12-35), respectively (Table 1, P < 0.001).In-hospital mortality was calculated using the chi-squared test and showed a statistically significant difference (Table 1, P = 0.04).The mortality rate for patients with a positive stool test was 28% compared to 25% for patients with a negative stool test.There was no statistically significant difference observed between the white blood cells (WBC) and platelets (Table 1, P > 0.05).There was also no statistical difference in the number of using RRT treatment between the two groups (Table 1, P > 0.05).Regarding the sources of infectious pathogens, there are certain statistical differences between gram-negative bacteria and gram-positive bacteria.There were statistical differences in the source of infection, but most of the infections in the two groups occurred in the respiratory and blood systems (Table1, P < 0.05).There was a statistically significant difference in the time it took to test for fecal pathogens between the two groups, with the PPS group taking an average of 6 days and the NPS group taking an average of 8 days (Table1, P < 0.001).

Impact of PPS on 90-Day survival, hospital survival, and 30-day survival
The KM curves demonstrate that mortality in patients with sepsis-associated AKI was similar between the NPS (48.45%) and PPS (52.93%) groups (P = 0.01; Fig. 1A).The 30-day cumulative mortality rate in the PPS group was 30.85%, which was higher than the 28.35% in the NPS group (P = 0.16; Fig. 1B).The probability of death during hospitalization in the PPS group (42.25%) was slightly higher than in the NPS group (43.69%;P = 0.21; Fig. 1C).The KM curves after PSM of hospital survival, 30-day survival, and 90-day survival are presented in Fig. 1D-F.Differences between the two groups after PSM did not alter our results.Enterobacteriaceae and Enterococci in stool samples were not associated with patient survival during hospitalization or with 30-day and 90-day survival.However, the presence of C. difficile in stool samples was significantly associated with decreased 90 days survival (Fig. 2A,B, Table 3).We performed causal inference of C. difficile positivity and 90-day prognosis, removed confounders, and drew a DAG diagram (Supplementary Fig. 1).After PSM analysis, the average treatment effect was 0.05 (95% CI 0.01-0.08,P < 0.05).This means that patients who develop C. difficile stool positivity have a 5% lower 90-day survival rate than those whose stool without C. difficile.
Variables that showed differences in 90-day patient survival were analyzed using single-factor Cox analysis with stool culture.Supplementary Table 1 demonstrates all variables initially considered in the forward stepwise model.The correlation between these factors and survival was statistically significant (Table 4).
Because treatment is already in using at the time of stool examination, antibiotics have an effect on fecal pathogens.The use of antibiotics was described in both groups (Table 5).In the group with positive pathogenic bacteria, vancomycin was used more frequently, with about 88% of patients using it, and there was a statistical difference between the two groups.Similarly, metronidazole was used more often in the PPS group and less often in the NPS group (P < 0.001).Penicillin/cephalosporin and quinolones were used less in the PPS group than in the NPS group (P < 0.05).There was no statistical significance in the antibiotic use of macrolides, sulfonamides, tetracyclines and aminoglycosides between the two groups (P > 0.05).

Discussion
In this study, we observed that the presence of C. difficile protists in stool samples was associated with a higher risk of in-hospital death, 30-day death, and 90-day death in patients with sepsis-associated AKI.PPS is associated with poor survival, particularly in those with C. difficile in their stool samples.To our knowledge, this study provides the first opportunity to investigate the exploratory role of PPS in patients with sepsis-associated AKI.
In this study, we observed that the presence of C. difficile and protists in stool samples significantly increased the risk of 90-day mortality.Protists were associated with worse in-hospital and 30-day survival.Multiple stool tests are necessary to avoid false negatives, so we did not include confounders in the number of tests 17 .Taking into account prolonged hospitalization with multiple fecal examinations, a worse 90-day survival was observed in PPS patients after propensity score matching to adjust for the possible influence of confounders.
C. difficile and protists in stool samples is associated with sepsis association AKI, and infections, and dysbiosis may negatively impact their survival 18,19 .This phenomenon also is due to increased levels of inflammatory cytokines and the fact that ischemia can lead to changes in the constituent organisms that make up the intestinal pathogens 20,21 .Sepsis can lead to a significant loss of microbial diversity in patients 22 .In our study, patients with elevated absolute neutrophil counts had lower survival rates than patients with decreased absolute neutrophil counts.Gastrointestinal colonization with C. difficile may be a potential risk factor for invasive fungal in sepsisassociated AKI patients.However, common dysbiosis pathogenic bacterial infections were not found in this study, like candidiasis.Therefore, patients with sepsis-associated AKI should be monitored for the presence of C. difficile in stool samples.www.nature.com/scientificreports/Patients whose stool samples contained C. difficile had worse survival outcomes than those with persistently NPS.In patients with sepsis-associated AKI, those whose intestines are colonized with antibiotic-resistant bacteria are likely to develop bacteremia.Inhibiting the intestinal flora can significantly reduce the incidence of bacteremia.High concentrations of C. difficile bacteria in feces can result in a high risk of bacterial translocation into the bloodstream.Microbial abundance and diversity in hemodialysis patients are on a downward trend.In addition, the mortality rate of sepsis-associated AKI patients undergoing further kidney replacement therapy is high, reaching up to 50% 23 .Broad-spectrum antibiotics increase the risk of Clostridium difficile infection.Therefore, reducing the use of high-risk antibacterial drugs associated with Clostridium difficile infection (such as second-and third-generation broad-spectrum cephalosporins, fluoroquinolones, clindamycin, etc.), as well as their frequency and duration, may improve patient clinical outcomes 24,25 .
Protists organisms constitute a heterogeneous category of eukaryotic microbes that bear a potential association with heightened mortality in individuals suffering from sepsis-related acute kidney injury 26     www.nature.com/scientificreports/prognosis may be due to the severe immune stress caused by protists 27 .For instance, protozoan infections can lead to endothelial dysfunction, hinder perturb the integrity of the glycocalyx layer as well as foster platelet aggregation and the initiation of coagulation cascades culminating in fibrin deposition 28 .Our results resonate with prior studies, affirming the contributory role of protists in augmenting patient fatality rates.
In the ICU, patients with sepsis association AKI have lower long-term 90-day survival.Our analysis showed that patients who were positive pathogens in stool had lower 90-day survival rates.This is somewhat related to treatment.Vancomycin, a potent antibiotic that primarily targets Gram-positive bacteria, is particularly effective against methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant staphylococci 29 .In the ICU, where patients with sepsis have PPS, the increased use of vancomycin may be indicative of efforts to control C. difficile infections rather than association with sepsis-associated acute kidney injury (AKI).In addition, our multivariate regression showed that it was associated with 90-day survival and also associated with quinolones.Quinolones are used less frequently in patients with positive stools, which may indicate the need for caution in clinical use.The source of the fungal infection also has a negative impact on the patient's prognosis for 90-day survival.Due to the patient's compromised immune function, it is difficult for the host to eliminate fungi, toxins, and metabolites 30,31 .The antibiotics used have a certain nephrotoxicity, resulting in a worse 90-day prognosis than in normal patients.
In the context of clinical implications, the detection of C. difficile and protists in stool samples, which are associated with adverse survival outcomes, indicates a recommendation for routine fecal microbiota screening in these patients, particularly during the early phase of hospitalization.Enhanced testing frequency minimizes false-negative results, enabling prompt identification and intervention in cases of potential microbial imbalance.In light of the observed association between antibiotic use and PPS, clinicians may wish to exercise caution when prescribing second-and third-generation cephalosporins, fluoroquinolones, clindamycin, and other high-risk antibiotics.It may be advisable to consider shorter courses where feasible.Treatment strategies should prioritize alternatives with reduced nephrotoxicity to safeguard both renal function and the integrity of the gut microbiome.www.nature.com/scientificreports/Maintaining microbial diversity and gut microbiome stability is vital to prevent bacterial translocation and secondary infections.Moreover, it is imperative to conduct large-scale, prospective, multi-center studies to validate these observations and elucidate the correlations between gut microbiota and clinical outcomes across diverse populations.These endeavors will facilitate the development of more precise therapeutic guidelines, tailored to the unique microbial profiles of patients with sepsis-associated acute kidney injury.A limitation of this study is its retrospective design coupled with a relatively small sample of bacterial species in the PPS group.Therefore, the role of microorganisms in fecal samples from patients with sepsis-associated AKI warrants further investigation.Second, this study was conducted with patients in the United States, where bacterial prevalence may vary.This variability could potentially influence the association between fecal microorganisms and clinical outcomes in other populations.Third, retrospective studies may have inherent flaws.For example, selection bias, included patients mainly collected stool after treatment, and treatment caused different causes of stool.Prospective studies are needed to investigate the correlation between the presence of stool pathogens at admission and prognosis.
In conclusion, our results suggest that PPS significantly influence the 90 days survival outcomes of patients with sepsis association AKI, particularly those with C. difficile in their stool samples.Depending on the differential detection of positive and negative antibiotics in stool samples, clinicians may find it beneficial to adjust their antimicrobial therapy regimens accordingly.

Patients
This study conducted a retrospective analysis of the MIMIC database, a comprehensive resource for intensive care patients 32 .The MIMIC database was established in 2003 through a collaborative effort involving the Beth Israel Deaconess Medical Center (BIDMC), MIT, the National Institutes of Health, Massachusetts General Hospital, emergency physicians, critical care physicians, computer scientists, and other critical care professionals.The current version, MIMIC-IV, includes patients admitted between 2008 and 2019.Prior to utilizing the database, our team completed the necessary training and obtained the appropriate certifications.As this project does not impact clinical care, all sensitive health information remains anonymous, thereby eliminating the need for individual patient consent.We followed the official MIMIC-IV tutorial to construct the research database using PostgreSQL (version 10.0, PostgreSQL Global Development Group).We extracted data from patients with sepsis-associated AKI who underwent comprehensive stool culture analysis for all bacteria and fungi in the gastrointestinal tract.The exclusion criteria for this study are as follows:1) Patients who are younger than 18 years old; 2) Patients with

Figure 1 .
Figure 1.KM Curve and after-PSM KM Curve Plot of sepsis-associated AKI patients on the basis of the results of stool cultures.A, D, 90 days survival of raw data and after PSM; B, E,30 days survival of raw data and after PSM; C, F, Hospital Survival of raw data and after PSM.

Figure 2 .
Figure 2. KM Curve of raw data and after PSM of 90 days Survival of sepsis-associated AKI patients on the basis of the results of C. difficile.

Table 1 .
. The poor Baseline characteristics between the two groups positive pathogens in stool and negative pathogens in stool.SOFA, Sequential organ failure assessment score; GCS, Glasgow coma scale; LOS, Length of Stay; ALT, Alanine transaminase; ALP, Alkaline phosphatase; AST, Aspartate aminotransferase; Spo2, Oxygen saturation.RRT, renal replacement therapy.

Table 4 .
Multivariate analysis of risk factors of 90 days Survival.

Table 5 .
Antibiotics use in PPS group and NPS group.